Introduction:
antimicrobial resistance is neglected in developing countries; associated with
limited surveillance and unregulated use of antimicrobials. Consequently, delayed
patient recoveries, deaths and further antimicrobial resistance occur. Recent
gastroenteritis outbreak at a children's home associated with multidrug resistant
non-typhoidal Salmonella spp, raised concerns about the magnitude of the
problem in Kenya, prompting antimicrobial resistance assessment preceding surveillance
system establishment.

Methods:
eight public medical laboratories were conveniently selected. Questionnaires
were administered to key informants to evaluate capacity, practice and utilization
of antimicrobial susceptibility tests. Retrospective review of laboratory
records determined antimicrobial resistance to isolates. Antimicrobial resistance
was
defined as resistance of a microorganism to an antimicrobial agent to which
it was previously sensitive and multidrug resistance as non-susceptibility
to at least one agent in three or more antimicrobial categories.

Conclusion:
antimicrobial susceptibility testing capacity was inadequate and the available
approved guidelines for this in Kenya were adhered to by one laboratory. The
resistance patterns indicated potential resistance to commonly prescribed antimicrobials.

Introduction

The problem of antimicrobial resistance (AMR) is major in resource-limited countries, mostly because of poverty [1, 2] and lack of proper surveillance systems. Surveillance of AMR generally provides data that is needed to raise the awareness of the problem and to implement necessary interventions [3]. Principally, laboratory-based surveillance is required for detection of resistance and monitoring for its spread [4]. Therefore, AMR patterns could very well be related to medical laboratories' ability and practices in performance of cultures and antimicrobial susceptibility testing (AST), and rationality of prescriptions by clinicians [3, 4]. In view of these, developing countries are being called upon to improve access to diagnostic laboratories, institute surveillance of emergence of resistance and regulate use of antibiotics besides instituting comprehensive national AMR policies and strategies [3]. Similarly, the increasing interconnection between countries and the globalization of trade and travel promotes the risk of importing bacteria or genes that could lead to the spread of AMR across borders and jeopardize effective treatment or prevention of bacterial infections [5]. Antimicrobial resistance being an emerging public health threat with local, national and global dimension has consequences ranging from delays in recovery to deaths from infectious diseases [6, 7] and further spread of resistance. As such, resistance to first-line drugs in most of the pathogens ranges from zero to almost 100% [4].

In Kenya, a recent outbreak of gastroenteritis at Mama Ngina Childrens' Home in December 2012 where multi- drug resistant (MDR) strain of non-typhoidal Salmonella spp was reported, in which two deaths occurred raised concerns about the magnitude of the problem in the country. This prompted the Ministry of Public Health and Sanitation to conduct rapid assessment to establish antimicrobial susceptibility testing capacity, practices and antimicrobial resistance patterns in selected clinical and public health laboratories. In determining the most appropriate indicator for AMR in the events that led to this study, focus was on enteric pathogens. Moreover, diarrhoeal diseases are a major cause of morbidity and mortality in developing countries, where bacteria is the most important pathogens in older children and adults, because of rampant empirical treatments in part contributed by inadequacies of laboratory services [8]. Consequently, the spread and gradual replacement of drug-sensitive strains of Salmonella typhi with multidrug-resistant strains that threatens to reduce clinical options for treating typhoid fever have been reported [9]. Evidence is also available from a study at Kenyatta National Hospital (KNH) indicating that the prevalence of Salmonella typhi resistant to two or more antimicrobials has been on the rise; from 50% in 1998 to 78% in 2004 [10]. These patterns are also likely to be observed with other enteric bacterial pathogens. In a bid to establish in-depth account of AMR and MDR in Kenya following the stated events, a rapid assessment was conducted in government owned medical laboratories.

Methods

Study sites: eight public medical laboratories consisting of two level
6, four level 5 and two level 4 health facilities were conveniently selected.
Level 6 was defined as national referral facility, level 5 as sub-national
facility and level 4 as district facility. These facilities were targeted
because of being high volume laboratories with better resources for bacterial
culture
and AST, therefore considered to be better placed in reporting AMR as
well as MDR.

Study design: rapid assessment; review of medical laboratory records
for
one year and assessment of laboratory capacity in AST using semi-standardized
forms and check list. Key informant interviews (KII) were conducted by interviewing
key laboratory personnel and clinicians in the facilities.

Definitions of antimicrobial resistance (AMR): antimicrobial resistance
was defined as resistance of a microorganism to an antimicrobial medicine to
which it was previously sensitive [11]. On the other hand,
MDR was defined as acquired non-susceptibility to at least one agent in three
or more antimicrobial categories [12].

Data collection: retrospective reviews of laboratory records on AST for
stool and blood cultures were carried out alongside KII. Data on culture and
AST from January to December 2012 was extracted from bacteriology registers using
semi-standardized forms. This was aimed at describing enteric isolates and determining
AMR patterns. Key informant interviews using semi-structured questionnaire for
which all affirmative responses would be verified were administered to bacteriology
section-heads of the respective medical laboratories and hospital clinicians.
This was to assess capacity of the laboratories to perform culture and AST, culture
and AST practices, and utilization of the laboratory culture and AST results
by clinicians. Data on culture media, reagents, typing sera, equipment, stock
organisms, infrastructure, quality assurance, archiving systems, outbreak preparedness,
use of Clinical and Laboratory Standards Institute (CLSI) or related standards,
turnaround time, and categories of antibiotic discs used were collected in the
process.

Data management and analysis: data from responses on KIIs and review
of bacteriology records was cleaned, entered in Epi Info and Microsoft Excel
2010
spread sheets from which counts, frequencies and tables were obtained. Aggregation
and data summaries were made for all laboratories assessed.

Ethical consideration: the permission to carry out the assessment was
granted by the Ministry of Health (MOH) and the medical superintendents of the
respective
health facilities where laboratories were located. To maintain confidentiality,
no patient identification information was extracted from the registers during
the review process. The findings were disseminated in a breakfast meeting attended
by MOH and line ministry officials among other stakeholders. Reports of the findings
were sent to all participating health facilities and other stakeholders. The
findings have also been presented in two international conferences; the 5th African
Field Epidemiology Network Scientific Conference and the 2nd joint Infection
Prevention Network Kenya (IPNET-K)/ Infection Control Africa Network (ICAN) conference.

Results

Facility description: between 10th and 28th February
2013, we assessed eight medical laboratories and analyzed data. All participating
facilities were high volume, public medical laboratories. They comprised
7(88%) clinical and 1(13%) public health laboratories, and all except
one were participating
in WHO/AFRO stepwise laboratory improvement scheme.

Laboratory capacity: all laboratories had basic capability to perform
bacterial culture and sensitivity tests for common bacterial pathogens; however,
only 1(13%)
laboratory had facilities for isolation of Campylobacter spp although
there were no reports of this organism being isolated. Despite the availability
of basic capacity to perform culture and sensitivity testing, 1(13%) laboratory
did not have any records and only 3(38%) performed blood cultures. Two (25%)
facilities had high throughput automated equipment (BactecTM machine)
for blood culture; however, both reported not performing blood cultures. All
(100%) laboratories reported not having service contracts for bacteriology equipment
and only 1(13%) had equipment validation reports. Six (75%) laboratories reported
having inadequate staff and lack of specialists in bacteriology and only 2(25%)
laboratories reported that all their staff had successfully undertaken competency
tests in culture and AST at the time of assessment. All the technicians from
7(88%) of the laboratories reported not having undergone any refresher training
in microbiology techniques in the past one year. However, all technicians from
1(13%) of the national reference laboratories reportedly participate in an in-house
continuous professional development sessions.

AST practices: overall, bacterial culture and AST practices varied in
all the eight laboratories assessed. There were 5(63%) laboratories with stool
sample
collection standard operating procedures (SOPs), 7(88%) with culture processing
SOPs and 5(63%) with AST SOPs. All (100%) laboratories had daily maintenance
reports for the essential microbiology equipments; however, 2(25%) laboratories
reported use of faulty microscopes. Installation reports of basic bacteriology
equipment were available in 5(63%) facilities. Varied blood culture bottles were
used in all the 3 laboratories performing blood culture. Each of the 3(38%) laboratories
that performed blood culture used different primary culture media, with one using
diphasic-Thioglycolate, another using diphasic- Haemolysin and another using
Brain-heart infusion. Blood sub-culture media in one national referral laboratory
comprised only of Chocolate Blood Agar (CBA) and Cystine Lactose Electrolyte
Deficient Agar (CLED). Laboratories are required to ensure standard depth of
media during culture, but only 1(13%) reported use of calibrated media dispenser.
Selenite F broth is an enrichment media essential for Salmonella spp isolation
in stool, but only 3(38%) of the laboratories were using it (Table
1). Stock of expired stool culture media and reagents were observed in
two laboratories.

Five (63%) facilities performed Internal Quality Controls (IQC) on media and reagents, while 3(38%) participated in microbiology External Quality Assurance (EQA), though not for culture and AST. Two (25%) laboratories consistently used Mueller Hinton media for AST, 3(38%) used single discs for AST, while 5 (63%) reported use of Mc Farland standard and stocked standard organisms for quality control (Table 1).
Overall, none of the assessed facilities had capability to characterize and identify pathogenic Escherichia coli, although reports of the organism were obtained in 4 (50%) of them. Six (75%) laboratories had typing sera for Vibrio cholera, 4 (50%) had typing sera for Salmonella spp and 2 (25%) had tying sera for Shigella spp. Salmonella and Shigella species were however reported by 7 (88%) laboratories without evidence of specific species serotyping (Table 1).

Antimicrobial resistance patterns: The following organisms showed AMR
patterns
to ampicillin: 8(80%) Shigella dysenteriae isolates, 3(75%) Shigella
sonnei isolates, 7(78%) Shigella boydii isolates and 24(90%) Salmonella spp
isolates. Similar resistance patterns were observed with tetracycline, in which16
(84%) Shigella flexineri isolates, 5(71%) Shigella boydii isolates
and 9(76%) Salmonella spp were obtained (Table
4). Resistance was observed with 5(100%) Shigella spp. isolates
to cotrimoxazole. Fifteen (91%) Salmonella spp. isolates also showed marked
resistance to cotrimoxazole. Similarly, Salmonella spp isolates showed
relatively high resistance to sulfamethoxazole, while absolute resistance to
the same antimicrobial agent was observed with 7(100%) isolates of Shigella
dysentriae , 4(100%) Shigella sonnei , 7(100%) Shigella boydii and
3(100%) Shigella flexineri . Two (100%) isolates of Salmonella spp
and 2(100%) of Shigella flexineri showed absolute resistance to amoxicillin.
High resistance to amoxicillin was also observed with 7(78%) isolates of Shigella
boydii . Twenty nine (90%) Escherichia
coli isolates were also resistant to ampicillin, 3(60%) to cotrimoxazole
and 21(60%) to cefotaxime. Multidrug resistance according to the definition adopted
in this study, might have occurred in cultures of Shigella flexineri and Salmonella
typhimurium isolates. Shigella flexineri exhibited MDR tendencies
where 3(100%) were resistant to cotrimoxazole, 3(100%) to sulfamethoxazole and
2(100%) to amoxicillin. Salmonella typhimurium on the other hand exhibited
MDR, with 6(100%) being resistant to ampicillin, 2(100%) to cotrimoxazole and
3(100%) to cefotaxime. However, it could not be ascertained whether the same
isolates of the two species were subjected across the three antimicrobial agents
(Table 4).

AST utilization: Ten clinicians were interviewed on utilization of AST
results for patient management. Eight (80%) of the clinicians reported not utilizing
laboratory AST results for patient management despite having knowledge of the
services being available at their hospital laboratories. The reasons for underutilization
of AST services by clinicians varied; all (100%) indicated that antibiotics tested
by the laboratories were not available in their hospital pharmacies. Three (30%)
blamed delays in AST laboratory results, 5(50%) blamed lack of feedback from
the laboratory and 2(20%) cited limited laboratory operation hours as the barriers
to their utilization of culture and AST services (Table
1).

Discussion

In the study to assess AMR, AST capacity and practices of medical laboratories, we found that commonly prescribed first-line antimicrobial agents are facing threat of resistance to common enteric pathogens in Kenya. Capacity of bacterial culture and AST in the assessed medical laboratories was inadequate. This undermines the quality and validity of results obtained from the laboratories. The available equipment for example, in all the laboratories assessed lacked validation reports and most lacked service contracts, posing a major quality challenge. Competence inadequacies were also evidenced by most laboratories reporting Escherichia coli, Salmonella and Shigella species without any evidence of serotyping.

Lack of use of certain essential media was also noted, with only three laboratories reporting use of Selenite F broth for isolation of Salmonella spp in stool culture, which reflected the inability of the other laboratories to isolate Salmonella spp especially when they are in low numbers. Mueller Hinton media and use of Mac Farland are very critical in AST among other quality processes. However, five of the eight laboratories used Mac Farland standards while only two consistently used Mueller Hinton media for AST. Stock of expired stool culture media and reagents were observed in two laboratories. Expired laboratory reagents when not removed from the shelves and disposed appropriately might tempt technicians to use them especially when experiencing stock outs. Use of expired reagents for testing leads to incorrect results. Cholera outbreak preparedness was evident in six facilities which had stock of both TCBS media and APW and five facilities which had stock transport media for sample transfer from the field. Five of the eight laboratories had stocked standard organisms for quality control. The rest of the laboratories could not effectively assure quality of the various culture media and reagents for lack of standard organisms.

We also found that bacterial culture practices were varied across the participating laboratories. For instance, blood culture bottles varied from use of conventional blood culture bottles as observed in one of the national level and one sub-national level laboratory to improvised blood culture bottles in another sub-national level laboratory. Improvised blood culture bottles might not be sterile thus a potential source of media contamination leading to false positive results and overall compromising quality of laboratory outcomes. Some laboratories lacked important SOPs for stool culture and AST. SOPs aims at standardizing the operations within the laboratory. Lack of approved SOPs for laboratory procedures will compromise the reliability and accuracy of the results performed by different technicians [13]. Other practices, as use of varied primary and sub-culture media for blood culture observed in the assessed laboratories, indicate lack of clear standards and guidelines in place. Clinical and Laboratory Standards Institute guidelines for AST were also only used in national level facilities; other laboratories lacked any standardized guidelines for antimicrobial susceptibility testing. A wide range of antibiotics was subjected to AST across most laboratories without reference to any approved guideline. This finding is inconsistent with the ideal practice in microbiology which defines that AST should be done according to CLSI [14] or any other approved guideline.

The results indicated that proportion of stool cultures with enteric bacterial pathogens isolated was 3.5%. This isolation rate is unlikely to be a true reflection of the distribution and occurrence of the pathogenic enteric bacteria in the population. During a 1-year period surveillance study in Western Kenya in which 729 stool specimens were collected from patients with diarrhea, 244 (33%) of the specimens yielded Shigella, Campylobacter, Salmonella, or Vibrio species [15]. This clearly indicates that the isolation rate of enteric pathogens in the assessed sites was low, occasioning concerns about laboratory capacity, practices and to some extent suspicion index by clinicians. Lack of enrichment media for stool cultures in four of the laboratories may have in part contributed to poor isolation rate. Other factors which might have contributed to low isolation rate of enteric bacteria by laboratories were: use of expired media and reagents, lack of internal quality controls (IQC) and standard organisms and poor processes in the pre-analytical phase like improper specimen collection, transport and delay in stool culture. However, AMR patterns, particularly of Salmonella typhimurium to cotrimoxazole, cefotaxime and ampicillin was consistent with those of other non-typhoidal Salmonellas described in Kenya by Kariuki et al [16].

The current AST practices as described in this document are largely resource wasting, undesirable in a resource-limited country, like Kenya. This assertion is supported by the fact that majority of the clinicians interviewed do not use laboratory AST results for patient management due barriers that can be avoided.
Limitations of the assessment: This study had a number of limitations which included convenient rather than random selection of health facilities, which essentially limits representativeness and generalizability. Other limitations include incompleteness of microbiology registers which made it difficult to conclusively measure AMR and MDR. Lack of standard practice in AST further complicated the ability to effectively compare antimicrobial agents in terms of resistance to the isolates, since there was no uniformity in antimicrobial agents subjected to specific organisms. Further, due to inadequacies in standards of isolation and characterization of bacterial agents from both stool and blood, it was not possible to authenticate the various isolates. Despite these limitations, the assessment may well represent the Kenyan situation in terms of antimicrobial resistance.

Conclusion

The basic equipment for AST which include microscopes, incubators, fridges and autoclaves were available in all the assessed laboratories. However, the practices and capacity for all these laboratories to perform bacterial culture and AST was deficient, which may support the observed low isolation rates. To a great extent, there were no approved standards being used in culture and AST, hence the patterns observed with the bacterial isolates and AMR/MDR may not be conclusive. However, the observations made may be a pointer to potential microbial resistance with commonly prescribed antimicrobials.
Recommendations: there is need to establish and strengthen capacity of microbiology laboratories in terms of technical skills, staffing, documents, mentorship, infrastructure, procurement, supplies (reagents, materials) and equipment.
There is need to standardize culture and antimicrobial susceptibility testing methods in medical laboratories in Kenya.
It is necessary to establish antimicrobial resistance surveillance system for common enteric pathogens.
There is need for study on policy, standards, guidelines and regulation to ensure appropriate antimicrobials use.

Competing interests

The authors declare no competing interests.

Authors’ contributions

Fredrick Odhiambo; Data collection, management and analysis, writing of the manuscript. Waqo Boru; Mentoring on the rapid assessment and Guidance on scientific writing. Arvelo Wences; Guidance on scientific writing. Onesmus Maina Muchemi, Everlyne Wambui Kanyina, Juliana Chepkemoi Tonui; Data collection, management and analysis. Samwel Amwai; Guidance on scientific writing. Zeinab Gura; Guidance on scientific writing. Tura Galgalo; Mentoring on the rapid assessment and Guidance on scientific writing. All authors have read and agreed to the final version of this manuscript and have equally contributed to its content and to the management of the case.

Acknowledgments

We thank all those who were in one way or another involved in the success of this assessment. In particular we would like to thank the East Africa Public Health Laboratory network for providing financial support to carry out the assessment. We acknowledge the FELTP-Kenya faculty and residents for their technical advice and moral support during data collection and scientific writing. We also acknowledge the management and staff of participating facilities for providing appropriate environment to conduct the assessment.